Project Summary Invasive aspergillosis (IA) is the most common filamentous fungal infection in immunocompromised patients. Mortality in patients diagnosed with this condition ranges from 70% to 90% at one year. Current methods to diagnose IA include imaging techniques and the detection of antigens, anti-Aspergillus antibodies, or Aspergillus nucleic acids. Unfortunately, current tests have only a poor to moderate accuracy for the diagnosis of invasive aspergillosis in immunocompromised patients. Here, we propose to develop highly sensitive and specific recombinant anti-aspergillus single-chain antibodies (scFvs) and using these as the basis for new and accurate diagnostic assays. In the R21 portion of this grant, our goal is to generate high affinity monomeric or multimeric aspergillus-specific scFvs and develop these reagents to the extent that their utility as potential diagnostics is demonstrated. In Aim 1, we will identify aspergillus- specific phage scFv clones by screening nave human scFv phage libraries. In Aim 2, we will identify scFvs that detect circulating aspergillus antigens. In Aim 3, we will express and purify these clones as monomeric scFvs and various multimeric constructs and validate their binding activities. In Aim 3, we will identify scFv pairs that can be used as capture and detection reagents for ELISA assays and diagnostic Ab arrays. In Aim 4, we will generate radiolabeled aspergillus-binding reagents and identify those with the highest affinity, stability, and target density. The R33 portion of this grant will involve three aspects. First, we will improve the sensitivity and specificity of our recombinant antibodies by generating and screening randomly mutated variants of these antibodies. Second, we will development these recombinant antibodies as serum diagnostics using either standard ELISA assays or antibody arrays. Third, we will develop methods to detect Aspergillus infection via the use of recombinant antibody imaging.